A COLORIMETRIC ASSAY FOR QUANTITATING BOVINE NEUTROPHIL BACTERICIDAL ACTIVITY

A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus. The procedure used the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate 13-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-gamma) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.